The present invention relates generally to molecular biology and enzymology and more specifically to nucleic acids encoding transporter proteins that regulate the transport of acetyl-coenzyme A (Ac-CoA) across membranes.
The structural diversity and complexity of sugar chains in membrane gangliosides are caused in part by the occurrence of several different species of sialic acid molecules. While N-acetylneuraminic acid and N-glycolylneuraminic acid are the most prevalent sialic acids in gangliosides in the brain, their O-acetylated forms are also found as minor components in LD1, B-series gangliosides including GD3 and GT1b, and C-series (NeuAca2-8NeuAca2-8NeuAca2-3Gal-R) gangliosides. Some biological properties are assumed to be associated with the modification of sialic acids by O-acetylation. For example, the expression of 9-O-acetylated gangliosides is apparently associated with neural cell differentiation and migration (Stallcup et al., Cold Spring Harbor Symp. Ouant. Biol., 48:761-773 (1983)). 9-O-acetylated GD3 detected by D1.1 antibody in rat brain was found to be localized in germinal cell zones but to disappear from postmitotic cells. It is absent in normal adult brain (Levine et al., J. Neuros., 4:820-831 (1984)). In the weaver mouse, however, the persistent expression of 9-O-acetylated gangliosides in the adult brain was associated with a defect in cerebellar granule cell migration (Johnstone et al., J. Neurochem., 51:1655-1657 (1988)). The attachment of an O-acetyl group in sialic acid residues causes significant effects on the enzymes of sialic acid metabolism such as sialidases. Effects are also seen on virus binding, cell adhesion, and the immunogenicity of sialic acid residues of gangliosides (for review, see Varki, Glycobiology, 2:25-40 (1992)).
In spite of its importance, the O-acetylation mechanism is poorly understood at the molecular and genetic levels. A series of work done by Varki""s group indicates that production of O-acetylated gangliosides is not a simple process, requiring the co-localization of the acceptor ganglioside GD3, acetyl-CoA (Ac-CoA) transporter, and acetyltransferase in the same Golgi compartment (Varki, Glycobiology, 2:25-40 (1992)). In fact, it was shown that detection of in vitro acetyltransferase activity was extremely difficult and the transfer activity was quickly lost, once intact cell membrane preparations were treated with detergents (Varki and Diaz, J. Biol. Chem., 260:6600-6608 (1985)).
Thus, a need exists to isolate and characterize other protein factors involved in the formation of O-acetylated gangliosides. The present invention satisfies this need and provides related advantages as well.
In accordance with the present invention, there are provided isolated mammalian Acetyl-Coenzyme A Transporter (AT) proteins. These AT proteins, or fragments thereof, are useful as immunogens for producing anti-AT antibodies, or in therapeutic compositions containing such proteins and/or antibodies. Invention AT proteins are also useful in bioassays to identify agonists and antagonists thereto.
In accordance with the present invention, there are also provided isolated nucleic acids encoding novel AT proteins. Further provided are vectors containing invention nucleic acids, probes that hybridize thereto, host cells transformed therewith, antisense oligonucleotides thereto and related compositions. The nucleic acid molecules described herein can be incorporated into a variety of recombinant expression systems known to those of skill in the art to readily produce isolated recombinant AT proteins. In addition, the nucleic acid molecules of the present invention are useful as probes for assaying for the presence and/or amount of a AT gene or mRNA transcript in a given sample. The nucleic acid molecules described herein, and oligonucleotide fragments thereof, are also useful as primers and/or templates in a PCR reaction for amplifying nucleic acids encoding AT proteins. Also provided are transgenic non-human mammals that express the invention protein.
Antibodies that are immunoreactive with invention AT proteins are also provided. These antibodies are useful in diagnostic assays to determine levels of AT proteins present in a given sample, e.g., tissue samples, Western blots, and the like. The antibodies can also be used to purify AT proteins from crude cell extracts and the like. Moreover, these antibodies are considered therapeutically useful to modulate the biological effect of AT proteins in vivo
Methods and diagnostic systems for determining the levels of AT protein in various tissue samples are also provided. These diagnostic methods can be used for monitoring the level of therapeutically administered AT protein or fragments thereof to facilitate the maintenance of therapeutically effective amounts. These diagnostic methods can also be used to diagnose physiological disorders that result from abnormal levels or abnormal structures of the AT protein.